Tuberculosis remains a devastating disease of mankind resulting in 3 million deaths per year. There are 10 million new cases of tuberculosis worldwide, and for the first time after a 32 year decline, the number of new cases of tuberculosis in the United States has increased for the last 3 years. In New York city alone, there was a 36% rise in the number of tuberculosis cases, predominantly in the black and hispanic populations. This rise of tuberculosis is most likely associated with the increase in AIDS patients and so is still likely to worsen. M. avium infections are a primary cause of fatality of AIDS patients. Although, M. tuberculosis, the causative agent of tuberculosis, is one of earliest described pathogens of man, analysis of this organism or any other mycobacterium by genetic means has been previously impossible. The goal of this proposal is to develop the systems and methodology, using modern recombinant DNA technology, to permit genetic analysis of the mycobacteria. By developing the first efficient transfection system and a novel E. coli-mycobacteria shuttle vector, we have introduced recombinant DNA molecules into both M. smegmatis and BCG vaccine strains for the first time. This novel vector, termed a shuttle plasmid, replicates in mycobacteria as a phage and in E. coli as a plasmid. This vector has been successfully used to introduce and stably express the first selectable marker gene for mycobacteria genetic research. We plan to use these genetic approaches to analyze the pathogenesis of mycobacteria, which could lead to new avenues for drug design. These methods will contribute to the development of BCG, currently the most widely used vaccine in the world, into a recombinant multivaccine vehicle, which would provide a novel approach to development of vaccines where T cell memory and effector responses are important.